A central step of gene expression is the transcription of DNA into RNA. The DNA-dependent RNA polymerase (RNAP) is the enzyme that catalyzes the formation of phosphodiester bonds to polymerize the RNA chain in all cellular organisms. In addition to the core RNAP protein, prokaryotes also require the sigma subunit, which binds core RNAP to form the holoenzyme, to achieve specificity of promoter recognition and initiation. When the holoenzyme interacts with promoter DNA to form the closed complex, a series of isomerization steps occur to yield the transcription-competent open complex, where the doublestranded DNA is melted over a 14 base pair region of DNA that includes the start site at +1. Extensive biochemical work by others indicates that region 2 of sigma plays a key role in the formation of the open complex. To further understand how the transition from closed to open promoter complex occurs and to specifically define the protein interactions between region 2 of sigma and DNA that are key to this transition, this research proposal is aimed at obtaining a high resolution structure (better than 3.5 Angstrom resolution) of the RNAP holoenzyme/promoter DNA complex.